- Email: [email protected]
The response of adrenergic neurones to axotomy and nerve growth factor
Brain Research, 94 (1975) 87-97
87
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
T H E R E S P O N S E OF A D R E N E R G I C NERVE GROWTH FACTOR
NEURONES
TO A X O T O M Y
AND
I. A. H E N D R Y
Department of Pharmacology, Australian National University, Canberra (Australia) (Accepted March 1 lth, 1975)
SUMMARY
Division of the axons of adrenergic neurones by crushing the postganglionic nerve trunks of rat superior cervical ganglia (SCG) at 6 days of age resulted in a permanent atrophy of the SCG reflected by a persistent decrease in the total protein content and in the activities of the enzymes tyrosine hydroxylase and D O P A decarboxylase. Administration of nerve growth factor ( N G F ) to rats with unilateral axotomy at a dose of 10 #g/g/day for the period 7-21 days of age resulted in hypertrophy of both normal and axotomised SCG. There was a progressive rise in the total protein content and in the activities of the two enzymes till the end of the treatment period in both SCG. After treatment ceased there was a progressive fall in the total protein content and activities of the two enzymes reaching a stable level after 4 weeks. The level reached for treated unoperated SCG remained elevated when compared to untreated control SCG. Axotomised treated SCG had approximately the same biochemical parameters as untreated control SCG and very much elevated over untreated axotomised SCG. These final levels persisted for at least 56 days after treatment had ceased. Animals showed a persistent ptosis after axotomy at 6 days of age but treatment with N G F resulted in a functional recovery by 11 weeks of age. It is suggested that there is normally a retrograde transfer of a factor during development from the target cell to the perikarya of the neurone permitting survival if the appropriate connections are made. Failure to make such a contact results in cell death. The cell death occurring normally, and the cell death resulting from axotomy, can both be prevented by N G F treatment leading to an hypertrophy of both SCG. This is consistent with the hypothesis that N G F is the retrograde trophic agent for the sympathetic nervous system in the developing animal.
88 INTRODUCTION
Previous results have shown that division of the postganglionic nerve trunks of the rat superior cervical gangl ion before the twelfth postnatal day results in a protbund atrophy of this ganglion 7. There is a decrease in the total protein content of the ganglion, as well as a decrease in the specific and total activities of two of the enzymes located within adrenergic neurones, tyrosine hydroxylase and DOPA decarboxylase. This response is no longer observed if axotomy is carried out after 3 weeks of age, when the adult response to axotomy occurs, resulting in chromatolysis and leading to regeneration. During the first 3 weeks after birth adrenergic nerve terminals reach the periphery and form a mature pattern, as assessed histologically as well as by the ability to accumulate noradrenaline12,13,zl,'e4. It therefore appears that during the critical period between the twelfth and twenty-first postnatal day adrenergic neurones must make contact with the peripheral target cells via intact axons in order to develop normally. This development apparently being regulated by some form of specific message originating from contact between the nerve terminals and target cells. The protein nerve growth factor (NGF) has been suggested as a retrograde trophic agentL This proposal explained the finding that a depot preparation of NGF bound to cellulose could replace an excised submaxillary gland so producing normal development of the superior cervical ganglion (SCG) innervating that gland 9. A retrograde axonal transport of NGF was then demonstrated11, and was shown to be specific for NGF in the adrenergic neurones of the sympathetic nervous system of rats 1°,28 and mice 1°. The present series of experiments examined the effect of NGF administration to animals after axotomy of the SCG. The results are consistent with the proposed role of NGF as a retrograde trophic agent. MATERIALS AND METHODS
Radiochemicals were obtained from the Radiochemical Centre (Amersham, U.K.): L-[side chain-2,3-aH]tyrosine (specific radioactivity 22 Ci/mmote)was diluted with non-radioactive L-tyrosine to a specific radioactivity of 6.7 Ci/mmole; L-[2,3aH]3(3,4-dihydroxyphenyl)alanine (specific radioactivity 3.6 Ci/mole). 6,7-Dimethyl5,6,7,8-tetrahydropteridine-HC1, B grade, was obtained from Calbiochem. (San Diego, Calif.); 2,5-diphenyloxazole (PPO) was obtained from Merck (Darmstadt, G.F.R.); Teric N8 from I.C.I. (Aust.); activated aluminium oxide, Brockman activity II from BDH Chemicals (Poole, U.K.) and Dowex 50X4 from Sigma (St. Louis, Mo.). Initial supplies of NGF were a kind gift from Dr. R. Stach and Prof. E. M. Shooter, subsequently NGF was purified from mouse submaxillary glands as the low molecular weight biologically active form of the protein, using the method of Schenker et ai. 'e2. Animals Six-day-old rats, from an outbred colony derived from the Wistar strain, were housed in litters of 8-10 with their mothers. Weaning was carried out at 4 weeks of age and the animals separated into sexes. Axotomy was performed on the right ganglion
89 TABLEI NUMBERS OF ANIMALS AT EACH TIME POINT
For each time point, 2 litters of 8-10 animals aged 6 days had axotomy performed on the right superior cervical ganglion. The next day surviving animals were divided into two random groups within each litter, one group receiving NGF and the other acting as the control group. Treatment was continued till 13 days of age for the animals killed on the 14th day, till 20 days of age for those killed on 21st day and the final dose was given on the 21st day of age for the remaining groups. At 28 days of age surviving animals were weaned and separated into sexes. Age (days)
Control
NGF treatment
7 14 21 28 35 48 77
6 6 8 6 9 6 6
-4 6 9 6 4 6
under ether anaesthesia with the aid of a stereoscopic dissecting microscope as previously described 7. Animals were treated by daily subcutaneous injection of either N G F in 155 m M NaC1 at a dose of 10/~g/g, or with vehicle alone from the day after the operation according to the outline in Table I. Animals were killed between 9-11 a.m. by exposure to ether vapour, and both SCG were completely freed from accompanying tissue. The number of animals killed at each time point are shown in Table I. E n z y m e assays Each ganglion was homogenized in 0.1 ml of 0.005 M Tris buffer, pH 7.5, containing 0.1 ~ Triton X-160. Duplicate samples of 10/zl were taken for each assay and total protein was estimated by the method of Lowry et al. 19. Reagent blanks in all assays consisted of 10/zl of 0.005 M Tris buffer, pH 7.5, containing 0.1 ~o Triton X-100. Radioactivity was determined in a Beckman scintillation counter in 2:1 xylene teric N8 containing 0.5 ~ x/v PPO scintillant at an efficiency of 21 ~ for tyrosine hydroxylase and 15 ~ for D O P A decarboxylase. Tyrosine hydroxylase activity was assayed as described by Hendry and Iversen s, using 7.5 /~M L-tyrosine and 720/~M 6,7-dimethyl-5,6,7,8-tetrahydropteridine. The reaction product [aH]DOPA was separated from unreacted tyrosine by adsorption on alumina columns and eluted with 2 ml 1 M acetic acid. D O P A decarboxylase was assayed as previously describedS,7, 20. The reaction product [3H]dopamine was separated from unreacted D O P A by separation on Dowex 50X4 (Na+). RESULTS
Ganglion size Axotomy at 6 days of age caused a long-lasting atrophy of the SCG when compared under the dissecting microscope with contralateral controls. Administration of
90
t •
/
0.L ,-.
/
4.
.4
• z~
\ \
"" M
",,.
/
i
-.
,
I
.
.<
o
;
0.t>
~
/
d
,,'
,
l 0..~
0.2
£ ,~
f
-
AXOTOMY+NGF
i.,,.,." ,..>,/
- .................
I~-~-~ • -"
AXOTOMY ~_....,4~.....°lj[l......
~b
Co
il-------. .....
3b
'
,~'o
-41
Ill . . . . . . . . . . . . . . . . . . . . . . .
~'0
,;o
zb
B'0
~GE IN DAYS 10-
/,/~\ /
Z
\
,"
o
O o,
L%
t
/
#
,'
.
],'
•
: ",.
i y¢ O
-..
----...<~ ~11~..
lb
"",u -''''~ ..... t 2'o
:J0
..................................... Y
.......... ab
CONTROt
~_ ............ 5o
go
:z;;;~r----~ 7'o
AGE fN DAYS
Fig. 1. A: effect of N G F on the tyrosine hydroxylase activity of normal and axotomised ganglia. Animals had both major postganglionic nerve trunks o f the right superior cervical ganglion erushed at 6 days after birth and the contralateral left ganglia served as the control. One group of animals was treated with N G F by daily injections of 10 #g/g from days 7 to 21 or until the day before sacrifice if killed before 21 days of age. All treatment ceased after the 21st day of age. B: effect of N G F on the specific activity of tyrosine hydroxylase in normal and axotomised ganglia. Values represent mean ± S.E.M. (veltical bars) and the number in each group is shown in Table I.
91 300 (
/ / / / / )
\
i
i
~9
,
[ ,'
200
z <
#t ,,
L < oc~ I? I00
-"m'" 1'0
2'0
......... 3'0
4'0
• ....... 5'0
: ................. 6'0
• 8'o
7'0
AGE IN DAYS
06
B
1
...........~,II~,
~
__-----co.,,o,
05
Z o
o~
",..r
E
t+..
....
.......I
o . . °. . , -
I Z
O2,
< 0
0
02-
0.1-
lb
2'0
50
4b
5b
go
zb
8b
AGE IN DAY3
Fig. 2. A: effect o f N G F o n the D O P A decarboxylase activity o f n o r m a l and a x o t o m i s e d ganglia. A n i m a l s were operated and treated as described for Fig. 1. B: effect o f N G F o n the specific activity of D O P A decarboxylase in n o r m a l and a x o t o m i s e d ganglia. Values represent m e a n ± S.E.M. and the n u m b e r in each group is s h o w n in Table I.
92 NGF caused the normal ganglia to become profoundly hypertrophied, and the ganglia remained enlarged for at least 8 weeks after cessation of treatment. NGF also caused an hypertrophy of axotomised ganglia; not only was the atrophy completely prevented but the size of the ganglia was similar to that found in contralateral unoperated NG F treated ganglia. These gross changes were reflected by the biochemical parameters.
Tyrosine hydroxylase Axotomy at 6 days of age resulted in a long-lasting decrease in the total activity of this enzyme in the SCG (Fig. I A). The administration of NGF from days 7 to 21 of age progressively increased levels of the enzyme in both normal and axotomised ganglia. At the end of this period enzyme levels in ganglia from NGF-treated animals exceeded those in normal untreated ganglia by a factor of 19 for unoperated ganglia and a factor of 12 for axotomised ganglia. At this time the enzyme activity in axotomised ganglia was 64 ~ 2 ~ for NGF-treated animals and 4 z~_-2 ~ for untreated animals relative to those in the contralateral intact ganglia (1(30~o). After treatment ceased at 3 weeks of age there was a progressive fall in enzyme levels in the treated unoperated ganglia, to approximately twice those in the untreated unoperated control, and to levels not significantly different to the control in the axotomised treated ganglia. These stable levels were reached by 4 weeks after treatment ceased, and at this stage the enzyme activity in axotomised ganglia was 23 ~ 7 ~ for untreated animals and 61 i 10 ~ for treated animals compared with contralateral intact controls (1(30~,). DOPA decarboxylase Levels of this enzyme also decreased after axotomy at 6 days of age (Fig, 2A). N GF increased the total activity of this enzyme in both axotomised and normal ganglia when administered daily from 7 to 21 days of age. Enzyme levels in the unoperated ganglia reached values 4.9 times normal, and those in axotomised ganglia were 3.9 times normal. At the end of the treatment period the enzyme activity in axotomised ganglia was 79 ± 12~o for the NGF-treated animals and 10 ~-± 5 ~ for untreated animals compared with contralateral intact controls (1(30~). After treatment was terminated at 3 weeks of age there was a fall in the enzyme levels and at 49 days there were 1.5 times normal for the unoperated ganglia and 0.9 times normal for the axotomised ganglia. By this time the enzyme activity in axotomised ganglia was 18 ~_ 4 ~ for untreated animals and 57 ± 8 ~ for animals treated with N G F compared with contralateral intact controls (100 ~,~). Protein content Axotomy at 6 days of age resulted in a complete failure of the normal development of the total protein content of the ganglia which remained at approximately the levels found in the ganglia at the time of operation (Fig. 3). N G F increased the total protein content of both normal and axotomised ganglia. Thus, after treatment for 721 days of age, the total protein was 4.5 times normal for unoperated ganglia and 3.7 times normal in axotomised ganglia. The protein content in axotomised ganglia was 79 -~ 8 ~ for NGF-treated, and 39 ± 7 % for untreated animals when compared with
93 500
/t\
400
300 Z
!
"-
Ii //
_z 200
......
-
,'
s
t
.__ .
"
CONTROL+NGF
"[
?
AXOTOMY+NG F CONTROL
........
100 -
t / J I~"
g'----Ib
_ ~
~
I .....
AXOTOMY
l ...... 2'0
• ..... 3"0
~i . . . . . . . . . . . 4'0
• ......................... 5'0
6'0
• 7'0
80
AGE IN DAYS
Fig. 3 Effect of NGF on the total protein content of normal and axotomised ganglia. Animals were operated and treated as described for Fig. 1. Values represent mean ~ S.E.M (vertical bars) and the number in each group is shown in Table I.
contralateral intact controls (1(30 ~o). As with the enzymes there was a rapid drop in the protein content which fell to an approximately constant level by 49 days of age, that is 4 weeks after treatment ceased. At this stage the protein content in axotomised ganglia was 70 ~_ 7 ~ for treated and 37 ± 4 ~o for untreated animals compared with contralateral intact controls (100 ~ ) .
Specific activity of tyrosine hydroxylase There was a small decrease in the specific activity of tyrosine hydroxylase from 14 days to adulthood in normal ganglia (Fig. 1B). Axotomy at 6 days of age markedly decreased the specific activity of this enzyme. The administration of N G F for 14 days postoperatively resulted in a large increase in the specific activity in both normal and axotomised ganglia. At the end of the treatment period unoperated gangliahad specific activities, 4 times that of normal ganglia. At this time the specific activity in operated ganglia was 13 4- 8 % for the untreated and 80 ~ 8 ~ for the treated animals compared with intact contralateral controls (1O0 ~ ) . As with the total activities the specific activity fell after treatment ceased, reaching a stable level 4 weeks later. The level of activity was 1.4 times normal for unoperated ganglia and 1.2 for the operated ganglia. At this time the specific activity in axotomised ganglia was 70 ± 20 % for untreated and 87 ~k 9 ~ for treated animals compared with intact contralateral controls
(100%). Specific activity of DOPA decarboxylase There was no change in the specific activity of D O P A decarboxylase in the normal ganglia from 21 to 77 days of age, and axotomy caused a significant reduction
94 in the specific activity of this enzyme (Fig. 2B). Treatment of animals with NG F for 7-21 days of life caused relatively little change in the specific activity of thi,~ enzyme, with a slight but insignificant (P > 0.5) reduction occurring over the treatment period. N G F increased the specific activity of the enzyme in axotomised ganglia, and the levels reached were not significantly different from unoperated ganglia treated with NGF. At the end of the treatment period the specific activity in the axotom~sed ganglia o/ was 38 ± 14/o for untreated and 86 :r: 17 ~ for NGF-treated animals compared with the contralateral intact controls (100 ~o). There was no change in the specific activity after cessation of treatment, and at 11 weeks of age the specific activity in axotomised / in relation to normal ganglia (100 O70) was 48 ± 16 ~ for untreated and 97 i 16 '2';,for treated animals. Effect on functional recovery
In the group of animals studied 70 days after axotomy, 4 of the 6 animals treated with 10 #g/g N G F for 14 days postoperatively showed no sign of the ptosis normally associated with division of the postganglionic nerve trunk. All of the 6 animals in the untreated group had evidence of ptosis. Effect o f different treatment periods
In order to compare the ability of N G F to prevent the effects of axotomy with the duration of the critical stage in the development of ganglia observed previously, animals were treated for various periods with 10/zg/g N G F , After axotomy at 6 days of age, animals were treated for the subsequent 3, 7 and 14 days and then left to grow until they were 35 days old. The activities of tyrosine hydroxylase and DOPA decarboxylase, as well as the total protein content of the ganglia, were then determined. As shown in Table II the longer the treatment period the more closely enzyme levels and protein content on the operated side approached those of the unoperated ganglia.
TABLE II EFFECT OF DIFFERENTTREATMENTPERIODSON GANGLIONPROTEIN AND ENZYMELEVELSAFTERAXOTOMY Animals had both major postganglionic nerve trunks of the superior cervical ganglion crushed on the right side at 6 days of age. Treatment of 10t~g/g N G F was given for the 3 periods shown and the animals left until they reached 35 days of age when they were sacrificed and the enzyme and protein activities determined as described in the text. Values represent mean ± S.E.M. for groups of 6 animals and are expressed as a percentage of the right ganglion compared to the unoperated left ganglion.
% Level of unoperated control at 35 days NGF treatment (days) Enzyme
Control
6-10
6-14
6-21
DOPA decarboxylase Tyrosine hydroxylase Total protein
26 ~ 8 25 = 7 43 ± 10
47 zk 7 36 :k 6 64 :k 8
52 ~ 10 41 ± 7 75 J: 5
68 = 15 81 ~: 10 83 i 12
95 DISCUSSION
Atrophy after axotomy at 6 days of age, and hypertrophy after the administration of N G F whether axotomy had been performed or not, were parallelled by changes in the total protein content of ganglia. The similar changes in the activities of the two enzymes studied, which are exclusively located within the adrenergic neurones '), suggest that these effects are predominantly produced by changes in the adrenergic neurones within the ganglion. The enzyme tyrosine hydroxylase is the rate limiting step in the synthesis of noradrenaline from tyrosine is, and has been shown to be under transsynaptic control during developmenO ,z5, as well as being influenced by the administration of N G F s,2s, z~. The present results confirm the profound effect of N G F on the increase in the activity of this enzyme in the normal ganglion, and, in addition, after axotomy there was a marked stimulation by N G F of the specific activity suggesting that N G F can replace the factor missing after the division of adrenergic axons. DOPA decarboxylase catalyses the second step in the synthesis of noradrenaline from tyrosine, and in the adult has been shown not to be regulated by transsynaptic induction 2,27. In addition, N G F increases the total activity of this enzyme with only minor changes in the specific activity 26. In the present experiments there was no significant rise in the specific activity of this enzyme after N G F treatment. Indeed there was a tendency for the specific activity of this enzyme to decrease. The present and previous 26 results suggest that N G F has no significant effect on the specific activity of this enzyme in the ganglia. The fall in the specific and total activity of DOPA decarboxylase resulting from axotomy probably reflects the number of surviving adrenergic cells, and this enzyme may be considered a non-specific marker for these neurones in the ganglion. The marked stimulation in the size 4, enzyme levels and total protein content of the ganglion by N G F declines after treatment ceases. Four weeks after treatment has been terminated stable levels of these parameters appear to have been reached. There is, however, some variation in the levels of these parameters between 48 and 77 days where there is an apparent increase for the NGF-treated control ganglia and a decrease in the NGF-treated axotomised ganglia. This apparent variation probably reflects variations between the litters used and perhaps small changes in the activity of the preparations of N G F that these two groups received. These results differ from previously reported work in mice 6 where treatment was carried out until day 14 and in that study 4 weeks after treatment ceased the tyrosine hydroxylase levels had returned to levels not significantly different from the normal. In the study reported here, treatment was continued throughout the critical period previously demonstrated 7, and this prolonged treatment resulted in the persistence of enlarged ganglia with increased enzyme levels and total protein content. This would further suggest that N G F is required throughout the critical period in order to obtain a survival of the neurones. The signal for the retrograde effects of axotomy on neuronal perikarya is not yet known 3. Several possibilities have been suggested, of which the two most likely are the loss of a postsynaptically derived trophic substance or the occurrence of a
96 normal repressor substance within the neurone which is lost after section of" the cell axon. In this regard horseradish peroxidase, a protein capable of limited retrograde axonal transport 15,16, is able to reach neuronal perikarya of the facial nucleus after injection around the site of axonal injury, before any signs of cell reaction are observed morphologically 14. This suggests that it is not the accumulation of any toxic materials from the site of axon section but rather the slower decrease of a normally occurring substance that is responsible for the neuronal cell body reaction. Of the two alternatives it would appear in the sympathetic nervous system to be the loss of the postsynaptically derived trophic agent which can adequately be replaced by N G P . The results presented here are consistent with the hypothesis. Thus, there is a critical period before which the adrenergic neurone must make contact via its axon with the target cell if development is to proceed normally. Failure of the neurone to contact the appropriate target cell, either during normal development or as a result of axotomy, results in the degeneration of the neurone. This requirement for contact with the target cell can be replaced by the administration of large amounts of exogenous N G F , and this treatment, if continued throughout the critical period, results in the permanent maintenance of neurones that would otherwise degenerate. This could explain the increase in the number of surviving mature neurones found after N G F administration in vi),olL These results add further support to the hypothesis that N G F acts as a retrograde trophic agent on adrenergic neurones causing them to reach a stable mature state after the appropriate synaptic contact has been made. ACKNOWLEDGEMENTS
The technical assistance of Miss J. Campbell is gratefully acknowledged, This work was carried out under a Queen Elizabeth II Fellowship. The author wishes to thank Prof. D. R. Curtis, Dr. A. W. Duggan and Dr. G. A. R, Johnston for criticism of the manuscript.
REFERENCES 1 BLACK, I. B., HENDRY, I. A., AND IVERSEN, L. L., Trans-synaptic regulation of growth and development of adrenergic neurons in mouse sympathetic ganglia, Brain Research, 34 (1971)
229-240. 2 BLACK,I. B., HENDRY,I. A., ANDIVERSEN,L. L., Differences in the regulation of tyrosine hydroxylase and dopa decarboxylase in sympathetic ganglia and adrenal, Nature New Biol., 231 (1971) 27-29. 3 CRAGO,B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 4 EDWARDS,D. C., FENTON, E. L., AND I-IENDRY,I. A., Quantitative aspects of the effects in mice of nerve growth factor and its antiserum. In E. ZAIMS(rEd.), Nerve Growth Factor and its Antiserum, Althone Press, London, 1972, pp. 237-252. 5 ["L~ANSON,R., ANDOWMAN,C. H., Pineal_dopa decarboxylase and monoamine oxidase activities as related to monoamine stores, J. Neurochem., 13 (1966) 597-605. 6 H~NDRV,I. A., Trans-synaptic regulation oftyrosine hydroxylase activity in a developing mouse sympathetic ganglion: effects of nerve growth factor (NGF), NGF antiserum and pempidine, Brain Research, 56 (1973) 313-320.
97 7 HENDRY, L A., The effects of axotomy on the development of the rat superior cervical ganglion, Brain Research, 90 (1975) 235-244. 8 HENDRY, I. A., AND IVERSEN, t . L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical ganglion, Brain Research, 29 (1971) 159-162. 9 HENDRY, I. A., AND IVERSEN, L. L., Changes in tissue and plasma concentrations of nerve growth factor following the removal of the submaxillary glands in adult mice and their effects on the sympathetic nervous system, Nature (Lond.), 243 (1973) 500-504. 10 HENDRY, I. A., STACK, R., AND HERRUP, K., Characteristics of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system, Brain Research, 82 (1974) 117-128. 11 HENDRY, I. A., STOCKEL, K., THOENEN, H., AND [VERSEN, L. L., The retrograde axonal transport of nerve growth factor, Brain Research, 68 (1974) 103-121. 12 IMAIZUMI, M., AND KUWAnARA, T., Development of the rat iris, Invest. Ophthal., 10 (1971) 733744. 13 IVERSEN, L. L., CHAMPLAIN, J. DE, GLOWINSK1, J., AND AXELROD, J., Uptake, storage and metabolism of norepinephrine in tissues of the developing rat, J. Pharmacol. exp. Ther., 157 (1967) 509-516. 14 KRISTENSSON, K., AND OLSSON, Y., Retrograde transport of horseradish peroxidase in transected axons. I. Fine relationship between transport and induction of chromatolysis, Brain Research, 79 (1974) 101-110. 15 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399406. 16 LAVAtL, J. H., AND LAVA1L, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 17 LEvI-MONTALCLNI, R., AND ANGELETTI, P. U., Nerve growth factor, Physiol. Rev., 48 (1969) 534569. 18 LEvirr, M., SPECTOR, S., SJOERDSMA, A., AND UDENFRIEND, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the profound guinea-pig heart, J. Pharmacol. exp. Ther., 148 (1965) 1-8. 19 LOWRY, O. H., ROSERROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 20 OESCH, F., OTTEN, V., AND THOENEN, n . , Relationship between the rate of axoplasmic transport and subcellular distribution of enzymes involved in the synthesis of norepinephrine, J. Neurochem., 20 (1973) 1691-1701. 21 OLSEN, L., Outgrowth of sympathetic adrenergic neurones in mice treated with nerve growth factor, Z. Zellforsch., 81 (1967) 155-173. 22 SCHENKER, A., MOBLEY, W. C., AND SHOOTER, E. M., Nerve growth factor. Rapid methods in its isolation and determination of its chain composition, J. biol. Chem., Submitted. 23 STOCKEL, K., PARAVICINI, U., AND THOENEN, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413-422. 24 TAMURA, T., AND SMELSER, G. K., Development of the sphincter and dilation muscles of the iris, Albrecht v. Graefes Arch. Ophthal., 89 (1973) 332 338. 25 THOENEN, H., Comparison between the effect of neuronal activity and nerve growth factor on the enzymes involved in the synthesis of norepinephrine, Pharmacol. Rev., 24 (1972) 255-267. 26 THOENEN, H., ANGELETTI, P. U., LEVI-MONTALCINI, R., AND KETTLER, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglion, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 1598-1602. 27 THOENEN, H., KETTLER, R., BURKORD, W., AND SANER, A., Neurally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit, NaunynSchmiedeberg's Arch. exp. Path. Pharmak., 270 (1971) 146-160.
87
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
T H E R E S P O N S E OF A D R E N E R G I C NERVE GROWTH FACTOR
NEURONES
TO A X O T O M Y
AND
I. A. H E N D R Y
Department of Pharmacology, Australian National University, Canberra (Australia) (Accepted March 1 lth, 1975)
SUMMARY
Division of the axons of adrenergic neurones by crushing the postganglionic nerve trunks of rat superior cervical ganglia (SCG) at 6 days of age resulted in a permanent atrophy of the SCG reflected by a persistent decrease in the total protein content and in the activities of the enzymes tyrosine hydroxylase and D O P A decarboxylase. Administration of nerve growth factor ( N G F ) to rats with unilateral axotomy at a dose of 10 #g/g/day for the period 7-21 days of age resulted in hypertrophy of both normal and axotomised SCG. There was a progressive rise in the total protein content and in the activities of the two enzymes till the end of the treatment period in both SCG. After treatment ceased there was a progressive fall in the total protein content and activities of the two enzymes reaching a stable level after 4 weeks. The level reached for treated unoperated SCG remained elevated when compared to untreated control SCG. Axotomised treated SCG had approximately the same biochemical parameters as untreated control SCG and very much elevated over untreated axotomised SCG. These final levels persisted for at least 56 days after treatment had ceased. Animals showed a persistent ptosis after axotomy at 6 days of age but treatment with N G F resulted in a functional recovery by 11 weeks of age. It is suggested that there is normally a retrograde transfer of a factor during development from the target cell to the perikarya of the neurone permitting survival if the appropriate connections are made. Failure to make such a contact results in cell death. The cell death occurring normally, and the cell death resulting from axotomy, can both be prevented by N G F treatment leading to an hypertrophy of both SCG. This is consistent with the hypothesis that N G F is the retrograde trophic agent for the sympathetic nervous system in the developing animal.
88 INTRODUCTION
Previous results have shown that division of the postganglionic nerve trunks of the rat superior cervical gangl ion before the twelfth postnatal day results in a protbund atrophy of this ganglion 7. There is a decrease in the total protein content of the ganglion, as well as a decrease in the specific and total activities of two of the enzymes located within adrenergic neurones, tyrosine hydroxylase and DOPA decarboxylase. This response is no longer observed if axotomy is carried out after 3 weeks of age, when the adult response to axotomy occurs, resulting in chromatolysis and leading to regeneration. During the first 3 weeks after birth adrenergic nerve terminals reach the periphery and form a mature pattern, as assessed histologically as well as by the ability to accumulate noradrenaline12,13,zl,'e4. It therefore appears that during the critical period between the twelfth and twenty-first postnatal day adrenergic neurones must make contact with the peripheral target cells via intact axons in order to develop normally. This development apparently being regulated by some form of specific message originating from contact between the nerve terminals and target cells. The protein nerve growth factor (NGF) has been suggested as a retrograde trophic agentL This proposal explained the finding that a depot preparation of NGF bound to cellulose could replace an excised submaxillary gland so producing normal development of the superior cervical ganglion (SCG) innervating that gland 9. A retrograde axonal transport of NGF was then demonstrated11, and was shown to be specific for NGF in the adrenergic neurones of the sympathetic nervous system of rats 1°,28 and mice 1°. The present series of experiments examined the effect of NGF administration to animals after axotomy of the SCG. The results are consistent with the proposed role of NGF as a retrograde trophic agent. MATERIALS AND METHODS
Radiochemicals were obtained from the Radiochemical Centre (Amersham, U.K.): L-[side chain-2,3-aH]tyrosine (specific radioactivity 22 Ci/mmote)was diluted with non-radioactive L-tyrosine to a specific radioactivity of 6.7 Ci/mmole; L-[2,3aH]3(3,4-dihydroxyphenyl)alanine (specific radioactivity 3.6 Ci/mole). 6,7-Dimethyl5,6,7,8-tetrahydropteridine-HC1, B grade, was obtained from Calbiochem. (San Diego, Calif.); 2,5-diphenyloxazole (PPO) was obtained from Merck (Darmstadt, G.F.R.); Teric N8 from I.C.I. (Aust.); activated aluminium oxide, Brockman activity II from BDH Chemicals (Poole, U.K.) and Dowex 50X4 from Sigma (St. Louis, Mo.). Initial supplies of NGF were a kind gift from Dr. R. Stach and Prof. E. M. Shooter, subsequently NGF was purified from mouse submaxillary glands as the low molecular weight biologically active form of the protein, using the method of Schenker et ai. 'e2. Animals Six-day-old rats, from an outbred colony derived from the Wistar strain, were housed in litters of 8-10 with their mothers. Weaning was carried out at 4 weeks of age and the animals separated into sexes. Axotomy was performed on the right ganglion
89 TABLEI NUMBERS OF ANIMALS AT EACH TIME POINT
For each time point, 2 litters of 8-10 animals aged 6 days had axotomy performed on the right superior cervical ganglion. The next day surviving animals were divided into two random groups within each litter, one group receiving NGF and the other acting as the control group. Treatment was continued till 13 days of age for the animals killed on the 14th day, till 20 days of age for those killed on 21st day and the final dose was given on the 21st day of age for the remaining groups. At 28 days of age surviving animals were weaned and separated into sexes. Age (days)
Control
NGF treatment
7 14 21 28 35 48 77
6 6 8 6 9 6 6
-4 6 9 6 4 6
under ether anaesthesia with the aid of a stereoscopic dissecting microscope as previously described 7. Animals were treated by daily subcutaneous injection of either N G F in 155 m M NaC1 at a dose of 10/~g/g, or with vehicle alone from the day after the operation according to the outline in Table I. Animals were killed between 9-11 a.m. by exposure to ether vapour, and both SCG were completely freed from accompanying tissue. The number of animals killed at each time point are shown in Table I. E n z y m e assays Each ganglion was homogenized in 0.1 ml of 0.005 M Tris buffer, pH 7.5, containing 0.1 ~ Triton X-160. Duplicate samples of 10/zl were taken for each assay and total protein was estimated by the method of Lowry et al. 19. Reagent blanks in all assays consisted of 10/zl of 0.005 M Tris buffer, pH 7.5, containing 0.1 ~o Triton X-100. Radioactivity was determined in a Beckman scintillation counter in 2:1 xylene teric N8 containing 0.5 ~ x/v PPO scintillant at an efficiency of 21 ~ for tyrosine hydroxylase and 15 ~ for D O P A decarboxylase. Tyrosine hydroxylase activity was assayed as described by Hendry and Iversen s, using 7.5 /~M L-tyrosine and 720/~M 6,7-dimethyl-5,6,7,8-tetrahydropteridine. The reaction product [aH]DOPA was separated from unreacted tyrosine by adsorption on alumina columns and eluted with 2 ml 1 M acetic acid. D O P A decarboxylase was assayed as previously describedS,7, 20. The reaction product [3H]dopamine was separated from unreacted D O P A by separation on Dowex 50X4 (Na+). RESULTS
Ganglion size Axotomy at 6 days of age caused a long-lasting atrophy of the SCG when compared under the dissecting microscope with contralateral controls. Administration of
90
t •
/
0.L ,-.
/
4.
.4
• z~
\ \
"" M
",,.
/
i
-.
,
I
.
.<
o
;
0.t>
~
/
d
,,'
,
l 0..~
0.2
£ ,~
f
-
AXOTOMY+NGF
i.,,.,." ,..>,/
- .................
I~-~-~ • -"
AXOTOMY ~_....,4~.....°lj[l......
~b
Co
il-------. .....
3b
'
,~'o
-41
Ill . . . . . . . . . . . . . . . . . . . . . . .
~'0
,;o
zb
B'0
~GE IN DAYS 10-
/,/~\ /
Z
\
,"
o
O o,
L%
t
/
#
,'
.
],'
•
: ",.
i y¢ O
-..
----...<~ ~11~..
lb
"",u -''''~ ..... t 2'o
:J0
..................................... Y
.......... ab
CONTROt
~_ ............ 5o
go
:z;;;~r----~ 7'o
AGE fN DAYS
Fig. 1. A: effect of N G F on the tyrosine hydroxylase activity of normal and axotomised ganglia. Animals had both major postganglionic nerve trunks o f the right superior cervical ganglion erushed at 6 days after birth and the contralateral left ganglia served as the control. One group of animals was treated with N G F by daily injections of 10 #g/g from days 7 to 21 or until the day before sacrifice if killed before 21 days of age. All treatment ceased after the 21st day of age. B: effect of N G F on the specific activity of tyrosine hydroxylase in normal and axotomised ganglia. Values represent mean ± S.E.M. (veltical bars) and the number in each group is shown in Table I.
91 300 (
/ / / / / )
\
i
i
~9
,
[ ,'
200
z <
#t ,,
L < oc~ I? I00
-"m'" 1'0
2'0
......... 3'0
4'0
• ....... 5'0
: ................. 6'0
• 8'o
7'0
AGE IN DAYS
06
B
1
...........~,II~,
~
__-----co.,,o,
05
Z o
o~
",..r
E
t+..
....
.......I
o . . °. . , -
I Z
O2,
< 0
0
02-
0.1-
lb
2'0
50
4b
5b
go
zb
8b
AGE IN DAY3
Fig. 2. A: effect o f N G F o n the D O P A decarboxylase activity o f n o r m a l and a x o t o m i s e d ganglia. A n i m a l s were operated and treated as described for Fig. 1. B: effect o f N G F o n the specific activity of D O P A decarboxylase in n o r m a l and a x o t o m i s e d ganglia. Values represent m e a n ± S.E.M. and the n u m b e r in each group is s h o w n in Table I.
92 NGF caused the normal ganglia to become profoundly hypertrophied, and the ganglia remained enlarged for at least 8 weeks after cessation of treatment. NGF also caused an hypertrophy of axotomised ganglia; not only was the atrophy completely prevented but the size of the ganglia was similar to that found in contralateral unoperated NG F treated ganglia. These gross changes were reflected by the biochemical parameters.
Tyrosine hydroxylase Axotomy at 6 days of age resulted in a long-lasting decrease in the total activity of this enzyme in the SCG (Fig. I A). The administration of NGF from days 7 to 21 of age progressively increased levels of the enzyme in both normal and axotomised ganglia. At the end of this period enzyme levels in ganglia from NGF-treated animals exceeded those in normal untreated ganglia by a factor of 19 for unoperated ganglia and a factor of 12 for axotomised ganglia. At this time the enzyme activity in axotomised ganglia was 64 ~ 2 ~ for NGF-treated animals and 4 z~_-2 ~ for untreated animals relative to those in the contralateral intact ganglia (1(30~o). After treatment ceased at 3 weeks of age there was a progressive fall in enzyme levels in the treated unoperated ganglia, to approximately twice those in the untreated unoperated control, and to levels not significantly different to the control in the axotomised treated ganglia. These stable levels were reached by 4 weeks after treatment ceased, and at this stage the enzyme activity in axotomised ganglia was 23 ~ 7 ~ for untreated animals and 61 i 10 ~ for treated animals compared with contralateral intact controls (1(30~,). DOPA decarboxylase Levels of this enzyme also decreased after axotomy at 6 days of age (Fig, 2A). N GF increased the total activity of this enzyme in both axotomised and normal ganglia when administered daily from 7 to 21 days of age. Enzyme levels in the unoperated ganglia reached values 4.9 times normal, and those in axotomised ganglia were 3.9 times normal. At the end of the treatment period the enzyme activity in axotomised ganglia was 79 ± 12~o for the NGF-treated animals and 10 ~-± 5 ~ for untreated animals compared with contralateral intact controls (1(30~). After treatment was terminated at 3 weeks of age there was a fall in the enzyme levels and at 49 days there were 1.5 times normal for the unoperated ganglia and 0.9 times normal for the axotomised ganglia. By this time the enzyme activity in axotomised ganglia was 18 ~_ 4 ~ for untreated animals and 57 ± 8 ~ for animals treated with N G F compared with contralateral intact controls (100 ~,~). Protein content Axotomy at 6 days of age resulted in a complete failure of the normal development of the total protein content of the ganglia which remained at approximately the levels found in the ganglia at the time of operation (Fig. 3). N G F increased the total protein content of both normal and axotomised ganglia. Thus, after treatment for 721 days of age, the total protein was 4.5 times normal for unoperated ganglia and 3.7 times normal in axotomised ganglia. The protein content in axotomised ganglia was 79 -~ 8 ~ for NGF-treated, and 39 ± 7 % for untreated animals when compared with
93 500
/t\
400
300 Z
!
"-
Ii //
_z 200
......
-
,'
s
t
.__ .
"
CONTROL+NGF
"[
?
AXOTOMY+NG F CONTROL
........
100 -
t / J I~"
g'----Ib
_ ~
~
I .....
AXOTOMY
l ...... 2'0
• ..... 3"0
~i . . . . . . . . . . . 4'0
• ......................... 5'0
6'0
• 7'0
80
AGE IN DAYS
Fig. 3 Effect of NGF on the total protein content of normal and axotomised ganglia. Animals were operated and treated as described for Fig. 1. Values represent mean ~ S.E.M (vertical bars) and the number in each group is shown in Table I.
contralateral intact controls (1(30 ~o). As with the enzymes there was a rapid drop in the protein content which fell to an approximately constant level by 49 days of age, that is 4 weeks after treatment ceased. At this stage the protein content in axotomised ganglia was 70 ~_ 7 ~ for treated and 37 ± 4 ~o for untreated animals compared with contralateral intact controls (100 ~ ) .
Specific activity of tyrosine hydroxylase There was a small decrease in the specific activity of tyrosine hydroxylase from 14 days to adulthood in normal ganglia (Fig. 1B). Axotomy at 6 days of age markedly decreased the specific activity of this enzyme. The administration of N G F for 14 days postoperatively resulted in a large increase in the specific activity in both normal and axotomised ganglia. At the end of the treatment period unoperated gangliahad specific activities, 4 times that of normal ganglia. At this time the specific activity in operated ganglia was 13 4- 8 % for the untreated and 80 ~ 8 ~ for the treated animals compared with intact contralateral controls (1O0 ~ ) . As with the total activities the specific activity fell after treatment ceased, reaching a stable level 4 weeks later. The level of activity was 1.4 times normal for unoperated ganglia and 1.2 for the operated ganglia. At this time the specific activity in axotomised ganglia was 70 ± 20 % for untreated and 87 ~k 9 ~ for treated animals compared with intact contralateral controls
(100%). Specific activity of DOPA decarboxylase There was no change in the specific activity of D O P A decarboxylase in the normal ganglia from 21 to 77 days of age, and axotomy caused a significant reduction
94 in the specific activity of this enzyme (Fig. 2B). Treatment of animals with NG F for 7-21 days of life caused relatively little change in the specific activity of thi,~ enzyme, with a slight but insignificant (P > 0.5) reduction occurring over the treatment period. N G F increased the specific activity of the enzyme in axotomised ganglia, and the levels reached were not significantly different from unoperated ganglia treated with NGF. At the end of the treatment period the specific activity in the axotom~sed ganglia o/ was 38 ± 14/o for untreated and 86 :r: 17 ~ for NGF-treated animals compared with the contralateral intact controls (100 ~o). There was no change in the specific activity after cessation of treatment, and at 11 weeks of age the specific activity in axotomised / in relation to normal ganglia (100 O70) was 48 ± 16 ~ for untreated and 97 i 16 '2';,for treated animals. Effect on functional recovery
In the group of animals studied 70 days after axotomy, 4 of the 6 animals treated with 10 #g/g N G F for 14 days postoperatively showed no sign of the ptosis normally associated with division of the postganglionic nerve trunk. All of the 6 animals in the untreated group had evidence of ptosis. Effect o f different treatment periods
In order to compare the ability of N G F to prevent the effects of axotomy with the duration of the critical stage in the development of ganglia observed previously, animals were treated for various periods with 10/zg/g N G F , After axotomy at 6 days of age, animals were treated for the subsequent 3, 7 and 14 days and then left to grow until they were 35 days old. The activities of tyrosine hydroxylase and DOPA decarboxylase, as well as the total protein content of the ganglia, were then determined. As shown in Table II the longer the treatment period the more closely enzyme levels and protein content on the operated side approached those of the unoperated ganglia.
TABLE II EFFECT OF DIFFERENTTREATMENTPERIODSON GANGLIONPROTEIN AND ENZYMELEVELSAFTERAXOTOMY Animals had both major postganglionic nerve trunks of the superior cervical ganglion crushed on the right side at 6 days of age. Treatment of 10t~g/g N G F was given for the 3 periods shown and the animals left until they reached 35 days of age when they were sacrificed and the enzyme and protein activities determined as described in the text. Values represent mean ± S.E.M. for groups of 6 animals and are expressed as a percentage of the right ganglion compared to the unoperated left ganglion.
% Level of unoperated control at 35 days NGF treatment (days) Enzyme
Control
6-10
6-14
6-21
DOPA decarboxylase Tyrosine hydroxylase Total protein
26 ~ 8 25 = 7 43 ± 10
47 zk 7 36 :k 6 64 :k 8
52 ~ 10 41 ± 7 75 J: 5
68 = 15 81 ~: 10 83 i 12
95 DISCUSSION
Atrophy after axotomy at 6 days of age, and hypertrophy after the administration of N G F whether axotomy had been performed or not, were parallelled by changes in the total protein content of ganglia. The similar changes in the activities of the two enzymes studied, which are exclusively located within the adrenergic neurones '), suggest that these effects are predominantly produced by changes in the adrenergic neurones within the ganglion. The enzyme tyrosine hydroxylase is the rate limiting step in the synthesis of noradrenaline from tyrosine is, and has been shown to be under transsynaptic control during developmenO ,z5, as well as being influenced by the administration of N G F s,2s, z~. The present results confirm the profound effect of N G F on the increase in the activity of this enzyme in the normal ganglion, and, in addition, after axotomy there was a marked stimulation by N G F of the specific activity suggesting that N G F can replace the factor missing after the division of adrenergic axons. DOPA decarboxylase catalyses the second step in the synthesis of noradrenaline from tyrosine, and in the adult has been shown not to be regulated by transsynaptic induction 2,27. In addition, N G F increases the total activity of this enzyme with only minor changes in the specific activity 26. In the present experiments there was no significant rise in the specific activity of this enzyme after N G F treatment. Indeed there was a tendency for the specific activity of this enzyme to decrease. The present and previous 26 results suggest that N G F has no significant effect on the specific activity of this enzyme in the ganglia. The fall in the specific and total activity of DOPA decarboxylase resulting from axotomy probably reflects the number of surviving adrenergic cells, and this enzyme may be considered a non-specific marker for these neurones in the ganglion. The marked stimulation in the size 4, enzyme levels and total protein content of the ganglion by N G F declines after treatment ceases. Four weeks after treatment has been terminated stable levels of these parameters appear to have been reached. There is, however, some variation in the levels of these parameters between 48 and 77 days where there is an apparent increase for the NGF-treated control ganglia and a decrease in the NGF-treated axotomised ganglia. This apparent variation probably reflects variations between the litters used and perhaps small changes in the activity of the preparations of N G F that these two groups received. These results differ from previously reported work in mice 6 where treatment was carried out until day 14 and in that study 4 weeks after treatment ceased the tyrosine hydroxylase levels had returned to levels not significantly different from the normal. In the study reported here, treatment was continued throughout the critical period previously demonstrated 7, and this prolonged treatment resulted in the persistence of enlarged ganglia with increased enzyme levels and total protein content. This would further suggest that N G F is required throughout the critical period in order to obtain a survival of the neurones. The signal for the retrograde effects of axotomy on neuronal perikarya is not yet known 3. Several possibilities have been suggested, of which the two most likely are the loss of a postsynaptically derived trophic substance or the occurrence of a
96 normal repressor substance within the neurone which is lost after section of" the cell axon. In this regard horseradish peroxidase, a protein capable of limited retrograde axonal transport 15,16, is able to reach neuronal perikarya of the facial nucleus after injection around the site of axonal injury, before any signs of cell reaction are observed morphologically 14. This suggests that it is not the accumulation of any toxic materials from the site of axon section but rather the slower decrease of a normally occurring substance that is responsible for the neuronal cell body reaction. Of the two alternatives it would appear in the sympathetic nervous system to be the loss of the postsynaptically derived trophic agent which can adequately be replaced by N G P . The results presented here are consistent with the hypothesis. Thus, there is a critical period before which the adrenergic neurone must make contact via its axon with the target cell if development is to proceed normally. Failure of the neurone to contact the appropriate target cell, either during normal development or as a result of axotomy, results in the degeneration of the neurone. This requirement for contact with the target cell can be replaced by the administration of large amounts of exogenous N G F , and this treatment, if continued throughout the critical period, results in the permanent maintenance of neurones that would otherwise degenerate. This could explain the increase in the number of surviving mature neurones found after N G F administration in vi),olL These results add further support to the hypothesis that N G F acts as a retrograde trophic agent on adrenergic neurones causing them to reach a stable mature state after the appropriate synaptic contact has been made. ACKNOWLEDGEMENTS
The technical assistance of Miss J. Campbell is gratefully acknowledged, This work was carried out under a Queen Elizabeth II Fellowship. The author wishes to thank Prof. D. R. Curtis, Dr. A. W. Duggan and Dr. G. A. R, Johnston for criticism of the manuscript.
REFERENCES 1 BLACK, I. B., HENDRY, I. A., AND IVERSEN, L. L., Trans-synaptic regulation of growth and development of adrenergic neurons in mouse sympathetic ganglia, Brain Research, 34 (1971)
229-240. 2 BLACK,I. B., HENDRY,I. A., ANDIVERSEN,L. L., Differences in the regulation of tyrosine hydroxylase and dopa decarboxylase in sympathetic ganglia and adrenal, Nature New Biol., 231 (1971) 27-29. 3 CRAGO,B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 4 EDWARDS,D. C., FENTON, E. L., AND I-IENDRY,I. A., Quantitative aspects of the effects in mice of nerve growth factor and its antiserum. In E. ZAIMS(rEd.), Nerve Growth Factor and its Antiserum, Althone Press, London, 1972, pp. 237-252. 5 ["L~ANSON,R., ANDOWMAN,C. H., Pineal_dopa decarboxylase and monoamine oxidase activities as related to monoamine stores, J. Neurochem., 13 (1966) 597-605. 6 H~NDRV,I. A., Trans-synaptic regulation oftyrosine hydroxylase activity in a developing mouse sympathetic ganglion: effects of nerve growth factor (NGF), NGF antiserum and pempidine, Brain Research, 56 (1973) 313-320.
97 7 HENDRY, L A., The effects of axotomy on the development of the rat superior cervical ganglion, Brain Research, 90 (1975) 235-244. 8 HENDRY, I. A., AND IVERSEN, t . L., Effect of nerve growth factor and its antiserum on tyrosine hydroxylase activity in mouse superior cervical ganglion, Brain Research, 29 (1971) 159-162. 9 HENDRY, I. A., AND IVERSEN, L. L., Changes in tissue and plasma concentrations of nerve growth factor following the removal of the submaxillary glands in adult mice and their effects on the sympathetic nervous system, Nature (Lond.), 243 (1973) 500-504. 10 HENDRY, I. A., STACK, R., AND HERRUP, K., Characteristics of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system, Brain Research, 82 (1974) 117-128. 11 HENDRY, I. A., STOCKEL, K., THOENEN, H., AND [VERSEN, L. L., The retrograde axonal transport of nerve growth factor, Brain Research, 68 (1974) 103-121. 12 IMAIZUMI, M., AND KUWAnARA, T., Development of the rat iris, Invest. Ophthal., 10 (1971) 733744. 13 IVERSEN, L. L., CHAMPLAIN, J. DE, GLOWINSK1, J., AND AXELROD, J., Uptake, storage and metabolism of norepinephrine in tissues of the developing rat, J. Pharmacol. exp. Ther., 157 (1967) 509-516. 14 KRISTENSSON, K., AND OLSSON, Y., Retrograde transport of horseradish peroxidase in transected axons. I. Fine relationship between transport and induction of chromatolysis, Brain Research, 79 (1974) 101-110. 15 KRISTENSSON, K., OLSSON, Y., AND SJOSTRAND, J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399406. 16 LAVAtL, J. H., AND LAVA1L, M. M., Retrograde axonal transport in the central nervous system, Science, 176 (1972) 1416-1417. 17 LEvI-MONTALCLNI, R., AND ANGELETTI, P. U., Nerve growth factor, Physiol. Rev., 48 (1969) 534569. 18 LEvirr, M., SPECTOR, S., SJOERDSMA, A., AND UDENFRIEND, S., Elucidation of the rate-limiting step in norepinephrine biosynthesis in the profound guinea-pig heart, J. Pharmacol. exp. Ther., 148 (1965) 1-8. 19 LOWRY, O. H., ROSERROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 20 OESCH, F., OTTEN, V., AND THOENEN, n . , Relationship between the rate of axoplasmic transport and subcellular distribution of enzymes involved in the synthesis of norepinephrine, J. Neurochem., 20 (1973) 1691-1701. 21 OLSEN, L., Outgrowth of sympathetic adrenergic neurones in mice treated with nerve growth factor, Z. Zellforsch., 81 (1967) 155-173. 22 SCHENKER, A., MOBLEY, W. C., AND SHOOTER, E. M., Nerve growth factor. Rapid methods in its isolation and determination of its chain composition, J. biol. Chem., Submitted. 23 STOCKEL, K., PARAVICINI, U., AND THOENEN, H., Specificity of the retrograde axonal transport of nerve growth factor, Brain Research, 76 (1974) 413-422. 24 TAMURA, T., AND SMELSER, G. K., Development of the sphincter and dilation muscles of the iris, Albrecht v. Graefes Arch. Ophthal., 89 (1973) 332 338. 25 THOENEN, H., Comparison between the effect of neuronal activity and nerve growth factor on the enzymes involved in the synthesis of norepinephrine, Pharmacol. Rev., 24 (1972) 255-267. 26 THOENEN, H., ANGELETTI, P. U., LEVI-MONTALCINI, R., AND KETTLER, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglion, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 1598-1602. 27 THOENEN, H., KETTLER, R., BURKORD, W., AND SANER, A., Neurally mediated control of enzymes involved in the synthesis of norepinephrine; are they regulated as an operational unit, NaunynSchmiedeberg's Arch. exp. Path. Pharmak., 270 (1971) 146-160.